Intermediates of peptide antagonists of neurotensin

ABSTRACT

The invention is directed to a novel compound of formula ##STR1## in which R is a hydroxyl, alkyloxy, phenylalkyloxy or --NH--CH 2  --COOH radical, R&#39; and R&#34; are identical and are each a hydroxy or methoxy radical and R&#39;&#34; represents a hydrogen, bromine, chlorine or iodine atom or a nitro radical. The invention is also directed to the salts of said compounds and their use.

The present invention relates to new compounds of formula: ##STR2## totheir salts, to their preparation and to their use.

In the formula (I), R represents a hydroxyl, alkyloxy, phenylalkyloxy or--NH--CH₂ --COOH radical, R' and R" are identical and each represent ahydroxyl or methoxy radical and R'" represents a hydrogen, bromine,chlorine or iodine atom or a nitro radical.

In the preceding and following definitions, the alkyl and alkyloxyradicals and portions contain 1 to 4 carbon atoms in a straight orbranched chain, Arg means arginine, Lys means lysine, Pro means proline,Fmoc means 9-fluorenylmethoxycarbonyl, Pmc means2,2,5,7,8-pentamethylchroman-6-sulphonyl and Boc meanstert-butoxycarbonyl.

The compound of formula (I) in which R, R' and R" represent a hydroxylradical and R'" represents a hydrogen atom can be prepared byfermentation of A9738 actinomycetes (CBS 162.94) and isolation of theproduct.

The fermentation is carried out according to conventional fermentationprocesses. A medium comprising peptone, a yeast extract, a meat extract,glucose, calcium carbonate, sodium chloride and agar is preferably usedas culture medium. This fermentation is preferably carried out at atemperature of between 25° and 30° C. The broth is then extracted andthe aqueous phase ultrafiltered and chromatographed a number of times.

The compounds of formula (I) in which R represents an alkyloxy orphenylalkyloxy radical can be prepared by esterification of acorresponding compound of formula (I) in which R represents a hydroxylradical.

This esterification is preferably carried out by reaction of an alcoholR--OH, in which R represents an alkyl or phenylalkyl radical, either inthe presence of thionyl chloride, according to the method described byM. E. Jung et al., Tetrahedron Letters, 30(32), 4211-4 (1989), or in thepresence of sulphuric acid or gaseous hydrochloric acid, at atemperature of between 0° and 25° C., or when R represents a benzyloxyradical, in the presence of para-toluenesulphonic acid, according to themethod described by T. E. Walker et al., J. Org. Chem., 51, 1175-9(1986). The tert-butyl ester can also be obtained by reaction withisobutene according to the process described in U.S. Pat. No. 3,496,219.

The compounds of formula (I) in which R represents an --NH--CH₂ --COOHradical can be prepared by reaction with glycine of a correspondingcompound of formula (I) in which R represents a hydroxyl radical.

It is particularly advantageous to use the compound of formula (I) inthe activated form. Mention may be made, as activated form, of thereaction product of the compound of formula (I) withN-hydroxysuccinimide, N-hydroxybenzotriazole or 2- 1H-benzotriazol-1-yl!-1,1,3,3-tetramethyluroniumhexafluoromethylphosphate, which can be prepared in an inert solvent,such as an amide such as dimethylformamide or N-methyl-2-pyrrolidone, achlorinated solvent such as chloroform or methylene chloride, an ethersuch as tetrahydrofuran or a mixture of these solvents, at a temperatureof between 15° and 60° C., in the presence of 4 Å molecular sieve or ofa coupling agent such as dicyclohexylcarbodiimide or alternatively apentafluorophenyl ester which can be prepared according to the methoddescribed by L. Kisfaludy et al., Synthesis, 325-327 (1983).

The condensation is generally carried out under the same temperature andreaction mixture conditions as those described above for the preparationof the activated form of the compound of formula (I), optionally in thepresence of pyridine or of an amine such as diisopropylethylamine. Thesereactions can be carried out without isolating the activated product.

The compounds of formula (I) in which R' and R" represent methoxyradicals can be prepared by reaction with trimethylsilyldiazomethane ofa corresponding compound of formula (I) in which R' and R" representhydroxyl radicals. When R represents an unprotected hydroxyl radical, acompound of formula (I) is obtained in which R, R' and R" are methoxyradicals.

This reaction is carried out in a lower aliphatic alcohol (methanol orethanol, for example) at a temperature in the region of 20° C.

The compounds of formula (I) in which R'" represents a nitro radical canbe prepared by nitration of a corresponding compound of formula (I) inwhich R'" represents a hydrogen atom.

This nitration is carried out by any known nitration method. Preferably,nitric acid is reacted, in acetic acid, at a temperature in the regionof 20° C.

The compounds of formula (I) in which R'" represents a chlorine atom canbe prepared by chlorination of a corresponding compound of formula (I)in which R'" represents a hydrogen atom.

This reaction is carried out by any known chlorination method.Preferably, 2,3,4,5,6,6-hexachloro-2,4-cyclohexadiene-1-one is reacted,in a dimethylformamide/carbon tetrachloride mixture, at a temperature inthe region of 20° C., according to the method described by M. Lemaire etal., Janssen Chimica Acta, 5(1), 3-8 (1987).

The compounds of formula (I) in which R'" represents a bromine atom canbe prepared by bromination of a corresponding compound of formula (I) inwhich R'" represents a hydrogen atom.

This reaction is carried out by any known bromination method.Preferably, bromine is reacted, in the presence of sodium acetate, inacetic acid, at a temperature in the region of 20° C.

The compounds of formula (I) in which R'" represents an iodine atom canbe prepared by iodination of a corresponding compound of formula (I) inwhich R'" represents a hydrogen atom.

This reaction is carried out by any known iodination method. Preferably,an alkali metal iodide (sodium iodide or potassium iodide, for example)is reacted, in the presence of a reagent such as1,2,4,6-tetrachloro-3α,6α-diphenylglycouril, in a low aliphatic alcoholsuch as methanol, at a temperature in the region of 20° C.

The compounds of formula (I) in which R represents a hydroxyl radicalcan optionally be converted to metal salts or to addition salts withnitrogenous bases according to methods known per se. These salts can beobtained by reaction, in a solvent, of a compound of formula (I) with ametal base (alkali metal or alkaline-earth metal, for example), ammonia,an amine or a salt of an organic acid. The salt formed is separated bythe usual methods.

The compounds of formula (I) can optionally be converted to additionsalts with an inorganic or organic acid by reaction with such an acid inan organic solvent, such as an alcohol, a ketone, an ether or achlorinated solvent.

Mention may be made, as examples of salts, of the addition salts withinorganic or organic acids (such as acetate, trifluoroacetate,propionate, succinate, benzoate, fumarate, maleate, oxalate,methanesulphonate, isethionate, theophyllinacetate, salicylate,methylenebis-β-oxynaphthoate, hydrochloride, sulphate, nitrate andphosphate), the salts with alkali metals (sodium, potassium or lithium)or with alkaline-earth metals (calcium or magnesium), the ammonium saltor the salts of nitrogenous bases (ethanolamine, trimethylamine,methylamine, piperidine, benzylamine, N-benzyl-α-phenethylamine,choline, arginine, leucine, lysine or N-methylglucamine).

The compounds of formula (I) are particularly advantageous asintermediates in the preparation of active compounds as neurotensinantagonists of formula: ##STR3## in which R represents a hydroxyl,alkyloxy, phenylalkyloxy or --NH--CH₂ --COOH radical, R₁ represents ahydrogen atom, an adamantylacetyl, adamantylcarbonyl, norbornylacetyl,norbornylphenoxycarbonyl, benzoyl, nicotinoyl, 4-phenylbenzoyl,4-tert-butylbenzoyl or 2-pyrrolidinecarbonyl radical or a protectivegroup for an amine functional group, R₂ represents an Arg or Lysresidue, R₃ represents an Arg or Lys residue, R₄ represents a Proresidue, m, n and p, which are identical or different, represent anumber equal to 0 or 1, it being understood that, when R₁ represents ahydrogen atom, the sum m+n+p is at least equal to 1, R₅ and R6 areidentical and represent a hydroxyl or methoxy radical and R₇ representsa hydrogen, chlorine, bromine or iodine atom or a nitro radical, andtheir equivalents in which the peptide bonds (CO--NH) between two aminoacid residues are replaced by --CH₂ --NH-- bonds and/or the peptide bond(CO--NH) between the R₂ and R₃ amino acid residues is replaced by a--CH═CH-- bond.

In the formula (II), each amino acid residue can be in the L or Dconfiguration.

Mention may be made, as protective group for the amine functional group,of the 9-fluorenylmethoxycarbonyl, tert-butylcarbonyl, acetyl, pivaloyland benzyloxycarbonyl groups, the phenyl in the benzyloxycarbonyl groupbeing optionally substituted by halogen, alkyl, alkyloxy or nitro.

The peptide compounds of formula (II) in which the sum m+n+p is at leastequal to 1 can be prepared by reaction of a compound of formula (I) witha derivative of formula:

    R.sub.1 --(R.sub.2).sub.m --(R.sub.3).sub.n --(R.sub.4).sub.p --OH (III)

in which R₁ represents an adamantylacetyl, adamantylcarbonyl,norbornylacetyl, norbornylphenoxycarbonyl, benzoyl, nicotinoyl,4-phenylbenzoyl, 4-tert-butylbenzoyl or 2-pyrrolidinecarbonyl radical ora protective group for an amine functional group, R₂, R₃, R₄, n, m and phave the same meanings as in the formula (II) and the sum m+n+p is atleast equal to 1, optionally followed by deprotection of the end aminefunctional group in order to obtain the compounds in which R1 representsa hydrogen atom and/or optionally, when R represents an alkyloxy orphenylalkyloxy radical, by deprotection of the carboxyl functional groupin order to obtain the compounds in which R represents a hydroxylradical.

Coupling of the derivatives of formulae (I) and (III) is carried out byany method known to a person skilled in the art for coupling an aminoderivative and a peptide.

It is particularly advantageous to use the derivative of formula (III)in the activated form. Mention may be made, as activated form, of thereaction product of the derivative of formula (III) withN-hydroxysuccinimide, N-hydroxybenzotriazole or 2- 1H-benzotriazol-1-yl!-1,1,3,3-tetramethyluroniumhexafluoromethylphosphate, which can be prepared in an inert solvent,such as an amide such as dimethylformamide or N-methyl-2-pyrrolidone, achlorinated solvent such as chloroform or methylene chloride, an ethersuch as tetrahydrofuran or a mixture of these solvents, at a temperatureof between 15° and 60° C., in the presence of 4A molecular sieve or of acoupling agent such as dicyclohexylcarbodiimide or alternatively apentafluorophenyl ester which can be prepared according to the methoddescribed by L. Kisfaludy et al., Synthesis, 325-327 (1983).

The condensation of the product of formula (I) with the activatedproduct of formula (III) is generally carried out under the sametemperature and reaction mixture conditions as those described above forthe preparation of the activated form of the derivative of formula(III), optionally in the presence of pyridine or of an amine such asdiisopropylethylamine. These reactions can be carried out withoutisolating the activated product.

It can be advantageous to carry out this reaction in the solid phase, ona resin. The preferred resins are4-hydroxymethylphenoxy-methyl/styrene-1% divinylbenzene copolymer (HMP,Applied Biosystems, Wang, J. Amer. Chem. Soc., 95, 1328, (1973)) orchlorotrityl chloride/polystyrene-1% divinylbenzene copolymer(Novabiochem) resins.

The optional deprotection of the end amine functional group can becarried out according to the usual techniques for the deprotection ofamines, such as those described by T. W. Greene, Protective Groups inOrganic Synthesis, John Wiley, New York. It is possible, for example, tocarry out the deprotection in an inert solvent such as dimethylformamideor a chlorinated solvent such as dichloromethane, in the presence ofpiperidine, at a temperature in the region of 20°-250° C.

The optional deprotection of the carboxyl functional group can becarried out by any method known to a person skilled in the art whichmakes it possible to change from a carboxylic ester functional group toa carboxyl functional group. Preferably, the deprotection is carried outin an inert solvent, such as dioxane, water or a mixture of thesesolvents, in the presence of lithium hydroxide, at a temperature in theregion of 0° C.

The derivatives of formula (III) are marketed or can be prepared byapplication or adaptation of the methods described by S. Doulut et al.,Peptide Research, 5(1), 30-38 (1992; J. Couder et al., Int. J. PeptideRes., 41, 181-184 (1993); V. K. Skukla et al., Can. J. Physiol.Pharmacol., 71, 211-216 (1993); M. H. Michael et al., J. Chem. Soc.Perkin I, 307-314 (1982); T. E. Christos et al., Bioorganic MedicinalChemistry Letters, 3(6), 1035-1040 (1993), in Patents WO 93/00359 and WO91/02750 and in the examples.

The compounds of formula (II) in which the sum m+n+p is at least equalto 1 and R₁ represents a hydrogen atom or a protective group for anamine functional group can also be prepared by reaction of thecorresponding derivative of formula (I) with successively an (R₄)_(p)--OH and/or (R₃)_(n) --OH and/or (R₂)_(m) --OH derivative in which R₄,R₃ and R₂ have the same meanings as in the formula (I), the aminoresidues of which are preferably protected, optionally followed bydeprotection.

This reaction is carried out under the conditions described in theliterature for the chemistry of peptides, such as those described abovefor the coupling of the derivatives of formula (I) and formula (III).

The compounds of formula (II) in which the sum m+n+p is equal to zeroand R₁ represents a protective group for the amine functional group canbe prepared by any method known to a person skilled in the art for theprotection of an amine functional group which does not modify theremainder of the molecule. In particular, when R₁ represents a9-fluorenylmethoxycarbonyl radical, a compound of formula (I) is reactedwith 9-fluorenylmethyl N-succinyl carbonate.

This reaction is generally carried out in an inert solvent, such asdioxane, in the presence of an aqueous sodium carbonate solution, at atemperature in the region of 20° C.

When R₁ represents a tert-butoxycarbonyl, acetyl, pivaloyl orbenzyloxycarbonyl radical in which the phenyl is optionally substitutedby halogen, alkyl, alkyloxy or nitro, a compound of formula (I) isreacted with a derivative R₁ --Cl in which R₁ represents atert-butoxycarbonyl, acetyl, pivaloyl or benzyloxycarbonyl radical inwhich the phenyl is optionally substituted by halogen, alkyl, alkyloxyor nitro.

This reaction is preferably carried out in an inert solvent, such asdimethylformamide, in the presence of a base, such as a trialkylamine(diisopropylethylamine, for example), at a temperature in the region of20° C.

The compounds of formula (II) in which m+n+p is equal to zero and R₁represents an adamantylacetyl, adamantylcarbonyl, norbornylacetyl,norbornylphenoxycarbonyl, benzoyl, nicotinoyl, 4-phenylbenzoyl,4-tert-butylbenzoyl or 2-pyrrolidinecarbonyl radical can be prepared byreaction of a compound of formula (I) with a chloride R₁ --Cl in whichR₁ represents an adamantylacetyl, adamantylcarbonyl, norbornylacetyl,norbornylphenoxycarbonyl, benzoyl, nicotinoyl, 4-phenylbenzoyl,4-tert-butylbenzoyl or 2-pyrrolidinecarbonyl radical.

This reaction is preferably carried out in an inert solvent, such asdimethylformamide, in the presence of a base, such as a trialkylamine(diisopropylethylamine, for example), at a temperature in the region of20° C.

The compounds of formula (II) can be purified by the usual knownmethods, for example by chromatography or extraction.

It is understood by a person skilled in the art that, for theimplementation of the processes described above, it may be necessary tointroduce protective groups for the amino functional groups in order toavoid side reactions, such as those groups described by T. W. Greene,Protective Groups in Organic Synthesis, John Wiley, New York.

The compounds of formula (II) in which R represents a hydroxyl radicalcan optionally be converted to metal salts or to addition salts withnitrogenous bases according to methods known per se. These salts can beobtained by reaction, in a solvent, of a compound of formula (II) with ametal base (alkali metal or alkaline-earth metal, for example), ammonia,an amine or a salt of an organic acid. The salt formed is separated bythe usual methods.

The compounds of formula (II) can optionally be converted to additionsalts with an inorganic or organic acid by reaction with such an acid inan organic solvent, such as an alcohol, a ketone, an ether or achlorinated solvent.

Mention may be made, as examples of pharmaceutically acceptable salts,of the addition salts with inorganic or organic acids (such as acetate,trifluoroacetate, propionate, succinate, benzoate, fumarate, maleate,oxalate, methanesulphonate, isethionate, theophyllinacetate, salicylate,methylenebis-β-oxynaphthoate, hydrochloride, sulphate, nitrate andphosphate), the salts with alkali metals (sodium, potassium or lithium)or with alkaline-earth metals (calcium or magnesium), the ammonium saltor the salts of nitrogenous bases (ethanolamine, trimethylamine,methylamine, piperidine, benzylamine, N-benzyl-ox-phenethylamine,choline, arginine, leucine, lysine or N-methylglucamine).

The compounds of formula (II) have advantageous pharmacologicalproperties. These compounds are antagonists of neurotensin and aretherefore useful in treating or preventing disorders associated withneurotensin.

Thus it is that these compounds can be used for the treatment orprevention of psychoses, anxious disorders, depression, cognitivedisorders, neurodegeneration, panic attacks, Parkinson's disease,Alzheimer's disease, schizophrenia, autism, tardive dyskinesia,irritable bowel syndrome, acute pancreatitis, ulcers, disorders ofintestinal motility, certain tumours sensitive to neurotensin, inweaning from chronic treatments and alcohol or drug abuse, allergic andinflammatory phenomena, cardiovascular and respiratory disorders andasthma.

The affinity of the compounds of formula (II) with respect toneurotensin was measured by their ability to displace the binding oftritiated neurotensin of its receptors present in a crude guinea pigcerebral cortex membrane preparation. The test is based on thatdescribed by M. Goedert et al., Brain Research, 304, 71-81 (1984): maleDunkin-Hartley guinea pigs (200-300 g) are sacrificed by decapitationand the brain quickly removed. All the following stages are carried outat 4° C. The cerebral cortex is dissected, weighed and homogenized in 5ml of Tris-HCl buffer (50 mM, pH 7.4) per gram of tissue with a polytron(force 6 for 15 seconds). The homogenate is centrifuged at 48,000 g for15 minutes and the pellet obtained washed twice in the same buffer. Thefinal pellet is homogenized in 3 ml of buffer per gram of startingtissue and retained in the form of aliquots (approximately 20 mg ofproteins per ml) at -80° C. until use. The protein content is measuredaccording to the method of Bradford, Anal. Biochem., 72, 248-254 (1976).The binding test is carried out in Tris-HCl 50 mM pH 7.4 buffercontaining 0.4 % of bovine serum albumin and 0.1 mM of bacitracin in thepresence of 0.15 mg of proteins per ml and 0.8 nM of ³ H!neurotensin for15 minutes at 25° C. The non-specific binding is determined in thepresence of 1 μM of neurotensin 1-13. The reaction is halted byfiltration and the radioactivity retained on the filter measured byscintillation counting. The products are studied over a range ofconcentrations in order to determine the 50 % inhibitory concentrationfor the specific binding. The compounds of formula (I) have, in thistest, an IC₅₀ of less than 15 μM.

The compounds of formula (II) have a low toxicity compatible with theiruse as an active principle in medicaments.

The following examples illustrate the invention.

A sample of the A9738 actinomycetes strain was deposited and registeredwith the Centraalbureau voor Schimmel culturen (CBS) at Baarn(Netherlands) under the conditions of the Treaty of Budapest, 22 Mar.1994, under the number CBS 162.94.

For the N.M.R. spectra, the nomenclature used is that of the ProteinData Bank, with the exception of the methyl radicals which are known asM. The amino acid residues of the macrocyclic residues of the compoundsof formulae (I) and (II) are known as X-TYR1 (N end amino acid), X-ILE2,X-TYR3 and X-TYR4 (C end amino acid). The amino acid residues of thechain are known as R, according to the name of the amino acid and itsposition in the chain.

EXAMPLE 1

Preparation of the compound of formula (I) in which R, R' and R"represent hydroxyl radicals and R'" represents a hydrogen atom:

A 250 ml medium containing 5 g/l of peptone, 5 g/l of yeast extract, 15g/l of glucose, 5 g/l of meat extract, 3 g/l of calcium carbonate, 5 g/lof sodium chloride and 1 g/l of agar is seeded, in a 2 liter Erlenmeyerflask, with a slant gelose of A9738 actinomycetes (CBS 162.94) in theform of a spore suspension. After agitating at 140 r/min in athermostatically controlled agitator at 28° C. for 72 hours, the wholeof the culture is transferred under sterile conditions to a primaryfermenter filled with 60 liters of the same medium. The fermenter isstirred at 300 r/min, aerated at 5 m³ /h and thermostatically controlledat 28° C. for 44 hours. The whole of the culture is then transferredunder sterile conditions to a production fermenter with 450 liters ofmedium sterilized for 40 minutes at 122° C. (distillers 5 g/l, beans 40g/l, glucose 5 g/l, soybean oil 10 g/l, sodium chloride 5 g/l and cobaltchloride 20 mg/l) and then maintained at a temperature of 28° C. for 94hours, with stirring at 250 r/min and aerated at the rate of 15 m³ /h.The broth (487 liters, pH 7.8) is then stirred with 5 % of clarcel,marketed by CECA, and then filtered in order to separate the myceliumfrom the filtrate. The filtrate (300 liters) is extracted with 2 times100 liters of ethyl acetate and the aqueous phase (250 liters) isultrafiltered through an ultrafiltration membrane whose cut-offthreshold is 20 kD, until a retentate volume of 40 liters is obtained.The ultrafiltrate (210 liters) is run through a stainless steel column(20×60 cm) containing 30 liters of Duolite S861 resin marketed by Rhomand Haas with a flow rate of 30 l/h. The column is rinsed with 80 litersof water and chromatography is then carried out by a stepwisemethanol/water gradient with a flow rate of 30 l/h and fractions of 10liters (50/50 by volume for Fractions 1 and 2, 60/40 by volume forFractions 3 to 8, 80/20 by volume for Fractions 9 and 10 and 100 %methanol for Fractions 11 and 12). Fractions 3 to 6 are combined, themethanol is removed under reduced pressure (3.4 kPa) and the aqueousphase is then lyophilized. 12.4 g of a yellow powder are thus obtained.This lyophilizate is disintegrated in 600 ml of methanol cooled to 40°C., the insoluble material is separated and the solution is concentratedunder reduced pressure (3.4 KPa) to give 4 g of dry extract. Thisextract is dissolved in 110 ml of a methanol/water solution (1/10 byvolume) and chromatographed on a stainless steel column (5×60 cm) filledwith Matrex C18-grafted silica (20 μ, 60 Å), marketed by Am icon, usinga Gilson system. The 50 ml fractions are collected with a Pharmaciafraction collector and the flow rate is 50 ml/minute. The gradientprofile used is the following: over 30 minutes, linear gradient from10/90 by volume methanol/water to 40/60 by volume methanol/water,stationary stage of 30 minutes at 40/60 by volume methanol/water andthen a linear gradient from 40/60 by volume methanol/water to 100%methanol over 40 minutes. Fractions 21 to 29 are combined andconcentrated under reduced pressure (3.4 kPa) to a volume of 10 ml. Thissolution is deposited at the top of a glass column (5×30 cm) filled withMacherey Nagel SC6 polyamide. Elution is carried out with water. 50fractions of variable volumes (20 to 70 ml) are collected. After havingset aside Fractions 1 to 12 (approximately 1100 ml), Fractions 13 to 25(600 ml) are combined and deposited on a glass column with a diameter of5 cm and containing 250 ml of DEAE Biogel A (Bio-Rad). The elution iscarried out with water. The first 900 ml of effluent and eluate are setaside and then the desired product is subsequently eluted with 2 litersof water. These 2 liters are run through an Analytichem Internationalcell with a volume of 75 ml (internal diameter 2.5 cm) containing 40 mlof 40 μ Bondesil C18-grafted silica marketed by AnalytichemInternational. The effluent is set aside and the product is eluted withmethanol. The methanolic solution is concentrated under reduced pressureto give 60 mg of pure product of formula (I) in which R, R' and R"represent hydroxyl radicals and R'" represents a hydrogen atom in theform of a slightly yellow powder IR spectrum: characteristic bands(cm⁻¹) at 3395 (phenol OH), 3300 (NH), 2970 (CH3), 2935 (CH2), 2875(CH3, CH2), 3125 to 2125 (acid OH), 1670 (acid and amide C═O), 1585 to1500 (benzene rings), 1235 (aromatic ether and phenol CO), 865 and 835(aromatic CH; Mass spectrum (Finengan TSQ46 in D/Cl with ammonia asreagent gas) M/z=634 MNH₄ ⁺, 617 MH⁺ ; N.M.R. spectrum (400 MHz, d₆-DMSO, δ in ppm): X-TYR1: broad NH2, HA 4.14, HB1, HB2 3.05, 3.20, HD17.10, HD2 6.80, HE1 6.81; X-ILE2: NH 8.38, HA 4.20, HB 1.58, MG1 0.88,HG21, HG22 1.20,1.51, MD 0.84; X-TYR3: NH 8.48, HA 3.63, HB1, HB2 2.42,2.88, HD1 6.60, HD2 5.59; X-TYR4: NH 4.22, HA 4.26, HB1, HB2 3.02, 3.47,HD1 7.24, HD2 7.26, HE1 7.46, HE2 6.78!.

EXAMPLE 2

Preparation of the compound of formula (I) in which R represents amethoxy radical, R' and R" represent hydroxyl radicals and R'"represents a hydrogen atom.

200 mg of the compound of formula (I) in which R, R' and R" representhydroxyl radicals and R'" represents a hydrogen atom are dissolved in 20ml of methanol and 0.8 ml of concentrated sulphuric acid is added. Thesolution is stirred for 18 hours at a temperature in the region of 20°C. 10 g of Duolite S 861 (Rhom and Haas) are then added and the mixtureis diluted with 200 ml of water and stirred for 30 minutes at atemperature in the region of 20° C. The resin is filtered and washed onsintered glass with 60 ml of water. The compound is then extracted byelution with 100 ml of methanol. After evaporation under reducedpressure (3.4 kPa), the residue is purified by chromatography on 170 mlof carboxymethyl-Triacryl resin (Sepracor), elution being carried outwith water and 10 ml fractions being collected. The fractions containingthe desired compound are combined and evaporated to dryness to give 40mg of compound of formula (I) in which R represents a methoxy radical,R'" represents a hydrogen atom and R' and R" represent hydroxyl radicalsin the form of a slightly yellow powder lonspray mass spectrum on SciexAPI III: M/z=631 (MH⁺); Infrared spectrum (FTIR), in methanol): 3390,3275, 2960, 2875, 1740, 1660, 1585+1500, 1510, 1215, 865+835 cm⁻¹ ; NMRspectrum (400 MHz, DMSO, Temp.=303K, δ in ppm): X-TYR1: NH2 7.92, HA4.78, HB1, HB2 3.01, 3.17, HD1 7.09, HD2 6.74, HE1 6.82, X-ILE2: NH8.63, HA 4.19, HB 1.62, MG1 0.82, HG21, HG22 1.18,1.48, MD 0.79; X-TYR3:NH 8.73, HA 3.58, HB1, HB2 2.42, 2.62, HD1 6.51, HD2 5.50; X-TYR4: NH4.22, HA 4.45, HB1, HB2 3.21, 3.39, HD1 7.12, HD2 7.36, HE1 7.58, HE26.82, OCH₃ 3.81!.

EXAMPLE 3

Preparation of the compound of formula (I) in which R, R' and R"represent hydroxyl radicals and R'" represents an iodine atom.

740 mg of the compound of formula (I) in which R, R' and R" representhydroxyl radicals and R'" represents a hydrogen atom are dissolved in 60ml of methanol and 180 mg of sodium iodide and then 133 mg of1,2,4,6-tetrachloro-3α, 6α-diphenylglycouril are added. The solution isstirred for 45 minutes at a temperature in the region of 20° C. Thesolvents are then evaporated under reduced pressure (2 kPa) and thecrude reaction mixture is fixed on 10 g of silica gel. This mixture isdeposited on a column with a diameter of 4 cm containing 150 g of silicagel and eluted with an ethyl acetate/acetic acid/water mixture(103/12/10 by volume), 100 ml fractions being collected. The fractionsbetween 900 and 2400 ml are combined and concentrated to dryness to give734 mg of an ochre-coloured solid. This solid is taken up in 20 ml ofwater and 100 ml of ethyl acetate, warmed to a temperature in the regionof 40° C. and then cooled to a temperature in the region of 200° C.,before being filtered and washed with 3 ml of water and then with threetimes 20 ml of ethyl acetate. It is dried under reduced pressure (30 Pa)at 400° C. in order to obtain 387.7 mg of compound of formula (I) inwhich R, R', R" represent hydroxyl radicals and R'" represents an iodineatom in the form of a beige-coloured solid melting at a temperatureabove 260° C. Mass spectrum on an Autospec VG Fisons, in LIMS (LiquidSecondary Ion Mass Spectroscopy), glycerol/thioglycerol matrix: M/z=743(MH)⁺ ; NMR spectrum (600 MHZ, DMSO, Temp. 303 K; chemical shifts inppm): X-TYR1: HA 3.98, HB1, HB2 3.16, 2.80, HD1, HD2 7.34, 6.67; X-ILE2:NH 8.51, HA 4.18, HB 1.62, MG1 0.84, HG21, HG22 1.49,1.16, MD 0.82;X-TYR3: NH 8.61, HA 3.64, HB1, HB2 2.66, 2.40, HD1, HD2 6.52, 5.29;X-TYR4: NH 4.19, HA 4.27, HB1, HB2 3.39, 3,02, HD1, HD2 7.40, 7.22, HE1,HE2 7.10, 6.70!.

EXAMPLE 4

Preparation of the compound of formula (I) in which R, R' and R"represent methoxy radicals and R'" represents a hydrogen atom.

20 ml of a 2M solution of trimethylsilyldiazomethane in hexane are addedover one hour, under an inert atmosphere, to a solution of 1.4 g of thecompound of the formula (I) in which R, R' and R" represent hydroxylradicals, R'" represents a hydrogen atom and R₁ --(R₂)_(m) --(R₃)_(n)--(R₄)_(p) represents Fmoc- dissolved in 30 ml of methanol and stirringis carried out for 18 hours at a temperature in the region of 200° C. Afurther 10 ml of a 2M solution of trimethylsilyldiazomethane in hexaneare added and stirring is carried out for 24 hours at a temperature inthe region of 200° C. 0.5 ml of acetic acid is added and the solventsare evaporated under reduced pressure (2 kPa). The residue obtained ispurified by chromatography on a column with a diameter of 4 cmcontaining 250 g of silica gel, elution being carried out successivelywith 750 ml of dichloromethane, 3000 ml of a dichloromethane/methanolmixture (98/2 by volume) and then a dichloromethane/methanol mixture(95/5 by volume), 100 ml fractions being collected. The fractionsbetween 2500 and 4400 ml are combined and evaporated to give 932 mg of awhite solid. The product can continue to be purified, by dissolving itin 10 ml of dichloromethane and by diluting with 50 ml of ethanol; thesolution is then concentrated under reduced pressure at a temperature inthe region of 200° C., until precipitation begins, and left standing for16 hours at a temperature in the region of +5° C. 441 mg of the compoundof formula (II) in which R, R' and R" represent methoxy radicals, R'"represents a hydrogen atom and R₁ --(R₂)_(m) --(R₃)_(n) --(R₄)_(p)represents Fmoc are thus obtained in the form of a white powder meltingat 145° C., with decomposition Mass spectrum on an Autospec VG Fisons,in LIMS (Liquid Secondary Ion Mass Spectroscopy), glycerol+thioglycerolmatrix: M/z=881 (MH)⁺ !. 0.1 ml of piperidine is added to 187 mg of thecompound obtained above dissolved in 20 ml of dichloromethane andstirring is carried out at a temperature in the region of 20° C. for 42hours. The solvents are evaporated under reduced pressure (2 kPa); theresidue is triturated in 10 ml of diethyl ether at a temperature in theregion of 35° C., cooled to a temperature in the region of 20° C.,filtered and washed three times in 5 ml of diethyl ether to give 140 mgof white powder which is taken up in 5 ml of water and extracted fivetimes with 20 ml of ethyl acetate. The solvents are evaporated underreduced pressure (2 kPa). Purification is continued by taking up in 1 mlof dichloromethane diluted with 10 ml of diethyl ether. 80 mg of thecompound of formula (I) in which R, R' and R" represent methoxy radicalsand R'" represents a hydrogen atom are thus obtained in the form of aslightly yellow powder melting at 2400° C., with decomposition Massspectrum on an Autospec VG Fisons, in LIMS (Liquid Secondary Ion MassSpectroscopy), glycerol+thioglycerol matrix: M/z=659 (MH)⁺ !.

EXAMPLES OF THE PREPARATION OF COMPOUNDS OF FORMULA (II): Example A

61 mg of the derivative of formula (I) in which R, R' and R" representhydroxyl radicals and R'" represents a hydrogen atom, 64 mg ofFmoc-Arg-Pro-OH and 13 mg of N-hydroxybenzotriazole are dissolved in 1ml of dimethylformamide containing 4A molecular sieve. After 15 hours,10 μl of pyridine and 21 mg of dicyclohexylcarbodiimide are added andthe mixture is left to react for 72 hours at 40° C. 15 ml of water arethen added and the precipitate thus formed is collected on sinteredglass No. 4. The solid is disintegrated in 10 ml of methanol and thenfiltered. The filtrate is diluted to 15 ml and then water (85 ml) isadded. The solution is subjected to high performance liquidchromatography with an octadecyl-grafted silica column (250×10 mm) at aflow rate of 3.5 ml/minute. The elution is carried out using a lineargradient from water containing 0.07% trifluoroacetic acid toacetonitrile/water (70/30 by volume) containing 0.07% trifluoroaceticacid. 1.7 ml fractions are collected. Those containing the desiredproduct are combined and evaporated under reduced pressure. 0.6 mg ofthe compound of formula (II) in which R represents a hydroxyl radical,R₁ --(R₂)_(m) --(R₃)_(n) --(R₄)_(p) represents Fmoc-Arg-Pro-, R₅ and R₆represent hydroxyl radicals and R₇ represents a hydrogen atom is thusisolated in the form of a beige powder Mass spectrum on a Perkin ElmerAPI III, in ionspray: M/z 1092 MH⁺ 1114 MNa⁺ ; N.M.R. spectrum (400 MHz,DMSO, δ in ppm): R-FMOC1: HA1, HA2 4.22, HB 4.30, aromatic protons 7.70,7.31, 7.40, 7.89; R-ARG2: NH 7.90, HA 4.20, HB1, HB2 1.45, 1.22, HG1,HG2 1.45, HD1, HD2 2.97, broad guanidine; R-PRO3: HA 4.42, HB1, HB21.83,1.88, HG1, HG2 1.88, 1.88, HD1, HD2 3.60, 3.40; X-TYR1: NH 6.98, HA4.69, HB1, HB2 2.92, HD1 6.90, HD2 6.70, HE1 6.68; X-ILE2: NH 8.27, HA4.05, HB 1.60, MG1 0.82, HG21, HG22 1.15, 1.55, MD 0.83; X-TYR3: NH8.65, HA 3.58, HB1, HB2 2.88, 2.40, HD1 6.57, HD2 5.47; X-TYR4: NH 4.27,HA 4.18, HB1, HB2 2.48, 2.52, HD1 7.16, HD2 7.31, HE1 7.51, HE2 6.78!.

The peptide Fmoc-Arg-Pro-OH can be synthesized in the solid phase, byusing an Fmoc synthesis strategy on an Applied Biosystems 431A deviceusing "standard Fmoc" cycles provided by the manufacturer withN-methyl-2-pyrrolidone as solvent. Deprotection of the α-aminefunctional groups is carried out with a 20% solution of piperidine inN-methyl-2-pyrrolidone for 20 minutes at each synthesis stage. Thepeptide is synthesized on 0.25 mmol of4-hydoxymethylphenoxymethyl/styrene-1% divinylbenzene copolymer (HMP,Applied Biosystems) Wang resin (J. Am. Chem. Soc., 95, 1328 (1973)). Thesymmetrical anhydride of Fmoc-Pro-OH is formed (1 mmol) by reaction for20 minutes with 0.5 mmol of dicyclohexylcarbodiimide in 1.3 ml ofN-methyl-2-pyrrolidone and 1.8 ml of dichloromethane. After reacting for13 minutes, 0.36 ml of 0.1M dimethylaminopyridine in dimethylformamideare added. After removal by filtration of the dicyclohexylurea formed,the symmetrical anhydride is reacted for 30 minutes with the resin. Theamine functional group of the proline is deprotected by reaction for 20minutes with a 20% solution of piperidine with N-methyl-2-pyrrolidone.The N-hydroxybenzotriazolyl ester of Fmoc-Arg(Pmc)-OH is formed byreaction of 1 mmol of Fmoc-Arg(Pmc)-OH in 4.1 ml ofN-methyl-2-pyrrolidone in the presence of 1 mmol ofN-hydroxybenzotriazol and 1 mmol of dicyclohexylcarbodiimide for 20minutes. After removal of the dicyclohexylurea formed, the ester isreacted for 30 minutes with the resin. A resin is thus obtained on whichis grafted the Fmoc-Arg(Pmc)-Pro group. The peptide is cleaved from theresin by treatment for 1 hour and 30 minutes with 10 ml oftrifluoroacetic acid, 0.75 g of phenol, 0.25 ml of ethanedithiol, 0.5 mlof thioanisole and 0.5 ml of water per 100 mg of peptidylresin. Afterremoval of the resin by filtration, the liquid phase is concentrated ona rotary evaporator for 30 minutes under reduced pressure (4 kPa). Thepeptide is then precipitated by addition of tert-butyl methyl ether andpetroleum ether (4/1 by volume) and recovered by centrifuging. Thepeptide is taken up in a minimum volume of trifluoroacetic acid,precipitated by addition of a mixture of tert-butyl methyl ether andpetroleum ether (4/1 by volume) and recovered by centrifuging, thisoperation is repeated twice. The peptide is then washed with 30 ml of amixture of tert-butyl methyl ether and petroleum ether (4/1 by volume),recovered by centrifuging and dried under vacuum (4 kPa). The peptide isused as is in the following stages of the synthesis.

Example B

A solution of 60 mg of Fmoc-Arg-Arg-Pro-OH, 16 mg ofN-hydroxysuccinimide and 20 mg of dicyclohexylcarbodiimide in 1.5 ml ofdimethylformamide is heated for 15 hours at 50° C. 50 mg of thederivative of formula (I) in which R, R' and R" represent hydroxylradicals and R'" represents a hydrogen atom and one drop of pyridine arethen added and the mixture is left to react for 72 hours at atemperature of 50° C. The reaction mixture is deposited at the top of asilica column (2.6×6 cm) equilibrated in dichloromethane. After washingthe dichloromethane (45 ml), the column is run through with an ethylacetate/acetic acid/water mixture (60/12/10 by volume). 4.5 ml fractionsare collected. The fractions containing the desired product are combinedand evaporated to dryness under reduced pressure (3.4 kPa) to give 10 mgof a beige powder. This powder is subjected to high performance liquidchromatography with an octadecyl-grafted silica column (250×10 mm) at aflow rate of 3.5 ml/minute. Elution is carried out using a lineargradient from water containing 0.07% of trifluoroacetic acid toacetonitrile/water (70/30 by volume) containing 0.07% of trifluoroaceticacid. 1.7 ml fractions are collected. Those containing the desiredproduct are combined and evaporated under reduced pressure to give 5 mgof the compound of formula (II) in which R represents a hydroxylradical, R₁ --(R₂)_(m) --(R₃)_(n) --(R₄)_(p) representsFmoc-Arg-Arg-Pro-, R₅ and R6 represent hydroxyl radicals and R₇represents a hydrogen atom in the form of a slightly beige powder Massspectrum on a Perkin Elmer API III, in ionspray: M/z: 1248 MH⁺, 624.8(M+2H)⁺⁺ ; N.M.R. Spectrum (400 MHz, DMSO, δ in ppm: R-Fmoc1: HA1, HA24.28, 4.32, HB 4.32, aromatic protons 7.68, 7.32, 7.42, 7.88; R-ARG2: NH7.30, HA 4.08, HB1, HB2 1.65, 1.50, HG1, HG2 1.50, HD1, HD2 3.15,guanidine 7.35, 6.90; R-ARG3: NH 7.98, HA 4.48, HB1, HB2 1.50, HG1, HG21.50, HD1, HD2 3.10, guanidine 7.22, 6.90; R-PRO4: HA 4.38, HB1, HB21.85,1.95, HG1, HG2 1.85,1.95, HD1, HD2 3.45, 3.61; X-TYR1: NH 6.90, HA4.92, HB1, HB2 2.98, HD1 6.90, HD2 6.90, HE1 6.80; X-ILE2: NH 8.12, HA4.13, HB 1.63, MG1 0.85, HG21, HG22 1.22, 1.51, MD 0.83; X-TYR3: NH8.48, HA 3.62, HB1, HB2 2.65, 2.70, HD1 6.58, HD2 5.52; X-TYR4- NH 4.22,HA 4.32, HB1, HB2 3.20, 3.35, HD1 7.28, HD2 7.30, HE1 7.51, HE2 6.78!.

The synthesis of the peptide Fmoc-Arg-Arg-Pro-OH is carried out asdescribed for the peptide Fmoc-Arg-Pro-OH in Example A. A resin on whicha group Fmoc-Arg(Pmc)-Pro has been grafted beforehand as described inExample A is deprotected from its Fmoc protective group with 20%piperidine in N-methyl-2-pyrrolidone for only 3 minutes in order torestrict formation of a corresponding diketopiperazine derivative. Theresin is washed copiously with N-methyl-2-pyrrolidone. In parallel, theN-hydroxybenzotriazolyl ester of Fmoc-Arg(Pmc)-OH is formed by reactionof 1 mmol of Fmoc-Arg(Pmc)-OH in 2.1 ml of N-methyl-2-pyrrolidone, 1 mlof 1M N-hydroxybenzotriazole in N-methyl-2-pyrrolidone and 1 ml of 1Mdicyclohexylcarbodiimide in N-methyl-2-pyrrolidone for 20 minutes. Afterremoval of the dicyclohexylurea formed, this ester is reacted for 30minutes with the grafted resin. A resin on which the groupFmoc-Arg(Pmc)-Arg(Pmc)-Pro is grafted is thus obtained. The peptideFmoc-Arg-Arg-Pro-OH is cleaved from the resin and purified in the sameway as Fmoc-Arg-Pro-OH.

Example C

7 mg of the product of Example B are dissolved in 180 μl ofdimethylformamide and 20 μl of piperidine are added. The solutionquickly becomes cloudy and a precipitate appears. The precipitate iscollected by centrifuging and washed with two times 200 μl ofacetonitrile. 1 mg of the compound of formula (II) in which R representsa hydroxyl radical, R₁ --(R₂)_(m) --(R₃)_(n) --(R₄)_(p) representsH-Arg-Arg-Pro-, R₅ and R₆ represent hydroxyl radicals and R₇ representsa hydrogen atom, in the form of the piperidine salt, is thus obtainedMass spectrum on a Perkin Elmer API III, in ionspray: M/z 1026 MH⁺ !.

Example D

50.3 mg of pentafluorophenyl N-Fmoc-L-prolinate are added to a solutionof 62 mg of compound of formula (I) in which R, R' and R" representhydroxyl radicals and R'" represents a hydrogen atom and 52.5 μl ofdiisopropylethylamine in 3 ml of dimethylformamide and stirring iscarried out for 18 hours at a temperature in the region of 25° C.Dilution is then carried out with 25 ml of ethyl acetate and 25 ml ofwater and acidification is then carried out with a 1 N aqueoushydrochloric acid solution. After separation by settling, the organicphase is washed with three times 25 ml of water and dried over magnesiumsulphate. After filtration, washing and evaporation of the filtrate (2kPa), 80 mg of solid are obtained. This solid is purified by highperformance liquid chromatography on a LiChroprep diol column (Merck)with a diameter of 2.5 cm and a length of 31 cm, the elution beingcarried out at a flow rate of 10 ml per minute, first with 200 ml of adichloromethane/ethanol mixture (95/5 by volume) and then 200 ml of adichloromethane/ethanol mixture (93/7 by volume). The fractionscontaining the expected compound are combined and evaporated to dryness(2 kPa) to give 25 mg of the compound of formula (II) in which Rrepresents a hydroxyl radical, R₁ --(R₂)_(m) --(R₃)_(n) --(R₄)_(p)represents Fmoc-Pro-, R₅ and R₆ represent hydroxyl radicals and R₇represents a hydrogen atom in the form of a white solid Mass spectrum(Autospec VG Fisons, in LSIMS): M/z=936 (M+H)⁺, M/z=714 (M-Fmoc+2H)⁺ ;Infrared spectrum (KBr): 3390, 2965, 2930, 2875, 3100 to 2250, 1680,1585, 1450, 1505, 835, 760 and 740 cm⁻¹ ; NMR spectrum (600 MHz, DMSO,Temp.=350 K, δ in ppm: R-Fmoc1: HA1, HA2 4.20, 4.32, HB 4.32, aromaticprotons 7.83, 7.28,7.48,7.88; R-PRO2: HA 4.22, HB1, HB2 1.74,2.08, HG1,HG2 1.78, 1.95, HD1, HD2 3.28, 3.35; X-TYR1: NH 7.62, HA 4.71, HB1, HB22.91, 3.06, HD1 6.89, HD2 6.84, HE1 6.68; X-ILE2- NH 7.95, HA 4.15, HB1.62, MG1 0.82, HG21, HG22 1.12,1.47, MD 0.79; X-TYR3: NH 8.24, HA 3.62,HB1, HB2 2.46, 2.70, HD1 6.62, HD2 5.52; X-TYR4: NH 4.22, HA 4.26, HB1,HB2 3.10, 3.38, HD1 7.18, HD2 7.25, HE1 7.45, HE2 6.78!.

Example E

9 mg of the compound of Example D are dissolved in 0.5 ml of adichloromethane/piperidine mixture (90/10 by volume). After 96 hours ata temperature in the region of 25° C., the solvents are evaporated underreduced pressure (2 kPa). The residue is taken up in 0.5 ml of water andthen washed with two times 1 ml of diethyl ether. The aqueous phase isbrought to a pH in the region of 4 with acetic acid. The suspensionobtained is then chromatographed on a preparative high performanceliquid chromatography column containing Bio-Rad 100 Å octadecyl-graftedsilica at a length of 25 cm and a diameter of 1 cm with a flow rate of 6ml per minute. Elution is carried out using a linear gradient fromwater/trifluoroacetic acid (100/0.07 by volume) towater/acetonitrile/trifluoroacetic acid (63/37/0.07 by volume), over 33minutes. 3 ml fractions are collected. The fractions containing thedesired product are combined, frozen to -80° C. and lyophilized (1 Pa)to give 0.6 mg of compound of formula (II) in which R₁ --(R₂)_(m)--(R₃)_(n) --(R₄)_(p) represents H-Pro-, R, R₅ and R₆ represent hydroxylradicals and R₇ represents a hydrogen atom in the form of a white solidMass spectrum (Finnegan TSQ46, in chemical ionization desorption withammonia as reagent gas): M/z=714 (MH⁺); NMR spectrum (600 MHz, DMSO,Temp.=303 K, δ in ppm): R-PRO1: HA 4.10, HB1, HB2 1.77,2.12, HG1, HG21.73, HD1, HD2 3.20; X-TYR1: NH 8.13, HA 4.78, HB1, HB2 2.94, 3.03, HD16.78, HD2 6.78, HE1 6.71; X-ILE2: NH 8.31, HA 4.10, HB 1.62, MG1 0.82,HG21, HG22 1.12,1.47, MD 0.79; X-TYR3: NH 8.66, HA 3.59, HB1, HB2 2.42,2.69, HD1 6.58, HD2 5.49; X-TYR4: NH 4.14, HA 4.31, HB1, HB2 3.10, 3.38,HD1 7.18, HD2 7.32, HE1 7.52, HE2 6.80!.

Example F

20 mg of compound of formula (I) in which R represents a methoxyradical, R' and R" represent hydroxyl radicals and R'" represents ahydrogen atom are added to a mixture of 23.2 mg of (Boc)₂ Lys-Ψ CH₂NH!-Lys(Boc)-Pro-OH trifluoroacetate, 11.4 mg of 2-1H-benzotriazol-1-yl!-1,1,3,3-tetramethyluroniumhexafluoromethylphosphate and 16 μl of diisopropylethylamine in 3 ml ofdimethylformamide at a temperature in the region of 20° C. The mixtureis stirred for 6 hours and 30 minutes at this same temperature. Thesolvents are evaporated under reduced pressure (15 Pa). The residue istaken up in 5 ml of dichloroethane and washed with two times 1 ml ofwater, two times 1 ml of a 0.1M sodium monophosphate solution, two times1 ml of a 0.25M sodium hydrogencarbonate solution and with two times 1ml of water. The organic phase is concentrated under reduced pressure(5.3 kPa). The residue is taken up in 2 ml of aqueous trifluoroaceticacid (95/5 by volume) at a temperature in the region of 20° C. for 1hour and 30 minutes. After concentrating to a volume of approximately0.2 ml (5.3 kPa), the product is precipitated by addition of methyltert-butyl ether and collected by centrifuging. 2.2 mg of compound offormula (II) in which R₁ --(R₂)_(m) --(R₃)_(n) --(R₄)_(p) representsH-Lys-Ψ CH₂ NH!-Lys-Pro-, R represents a methoxy radical, R₅ and R6represent hydroxyl radicals and R₇ represents a hydrogen atom are thusobtained with a pale yellow colour. Mass spectrum on Sciex API III, inionspray: M/z=970 (MH⁺)!

The compound (Boc)₂ Lys-Ψ CH₂ NH!-Lys(Boc)-Pro-OH can be preparedaccording to the method of S. Doulut et al., Peptide Research, 5(1),30-38 (1992), by reacting (Boc)₂ Lysinal with H-Lys(Boc)-Pro-OBg andthen by saponifying the end ester (OBg ester=N-benzhydrylglycolamideester: Tetrahedron, 44, 5101-5108 (1988)).

Example G

37 mg of compound as described in Example F are dissolved in 0.6 ml ofdioxane, 0.3 ml of water and 0.3 ml of 1M lithium hydroxide solution ata temperature in the region of 0° C. After stirring for 70 minutes at atemperature in the region of 0° C., dilution is carried out with 10 mlof aqueous acetic acid (90/10 by volume) and the crude product isinjected onto a preparative high performance liquid chromatographycolumn containing Bio-Rad RSL 100 Å octadecyl-grafted silica with alength of 30 cm and a diameter of 1 cm. Elution is carried out using alinear gradient from water/trifluoroacetic acid (100/0.07 by volume) towater/acetonitrile/trifluoroacetic acid (65/35/0.07 by volume), over 30minutes. The fractions containing the desired product are combined,frozen to -80° C. and lyophilized (1 Pa) to give 4.4 mg of the compoundof formula (II) in which R₁ --(R₂)_(m) --(R₃)_(n) --(R₄)_(p) representsH-Lys-Ψ CH2NH!-Lys-Pro-, R, R₅ and R₆ represent hydroxyl radicals and R7represents a hydrogen atom in the form of a white powder Mass spectrumon Sciex API III, in ionspray: M/z=956 (MH⁺)!.

Example H

The solid-phase synthesis is carried out using the equipment from thecompany Shandon (Life Science International Group), with the exceptionof the rotator for haemolysis tubes. The resin is confined in 3 mlhigh-density polyethylene (HDPE) syringes for solid-phase extractionequipped with teflon filters. These syringes are fitted to a two-wayteflon valve and are closed by a disposable HDPE finned stopper.Agitation of the syringes is carried out on a rotator for haemolysistubes. The washing and filtration operations are carried out on a workstation for solid-phase extraction (vacuum chamber equipped with Luerfittings, Shandon).

The synthesis is carried out on 50 μmol of resin in Fmoc chemistry. Thecouplings of the amino acids are carried out by treating the resin for 1hour with 250 μmol of the suitably protected amino acid in the presenceof 250 μmol of 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate, 250 μmol of N-hydroxybenzotriazole and 750 μmol ofdiisopropylethylamine in 1.2 ml of anN-methyl-2-pyrrolidone-dimethylformamide mixture (1/1 by volume).Deprotection of the Fmoc group is carried out by 3 successive treatmentsof the resin, for 1 minute, 1 minute and 20 minutes respectively, with 2ml of a 20% by volume solution of piperidine in N-methyl-2-pyrrolidone.

50 μmol of Fmoc-Gly-resin Wang resin! (Wang et al., J. Amer. Chem. Soc.,95, 1328 (1973)) are subjected successively to the following treatments:

Deprotection of the Fmoc group,

Washing in 5 times 2 ml of N-methyl-2-pyrrolidone,

Coupling of 2 equivalents of the compound of formula (I) in which R, R'and R" each represent a hydroxyl radical and R'" represents a hydrogenatom, the amino of which is protected by Fmoc,

Washing with 5 times 2 ml of N-methyl-2-pyrrolidone,

Deprotection of the Fmoc group,

Washing with 5 times 2 ml of N-methyl-2-pyrrolidone,

Coupling of 5 equivalents of Fmoc-proline,

Washing with 5 times 2 ml of N-methyl-2-pyrrolidone,

Deprotection of the Fmoc group,

Washing with 5 times 2 ml of N-methyl-2-pyrrolidone,

Coupling of 5 equivalents of Fmoc-arginine(Pmc),

Washing with 5 times 2 ml of N-methyl-2-pyrrolidone,

Deprotection of the Fmoc group,

Washing with 5 times 2 ml of N-methyl-2-pyrrolidone,

Coupling of 5 equivalents of Fmoc-arginine(Pmc),

Washing with 5 times 2 ml of N-methyl-2-pyrrolidone, and the peptide isthen cleaved by reaction in 10 ml of a trifluoroaceticacid/phenol/ethanedithiol/thioanisole/water mixture (4013111212 byvolume) for 90 minutes. The resin is removed by filtration. The filtrateis concentrated under reduced pressure using a rotary evaporatorequipped with a membrane pump and a dry ice trap for 1 hour, thetemperature of the bath being maintained at 45° C. The final volume ofthe concentrate is approximately 1 ml. The product is precipitated byaddition of 15 ml of methyl tert-butyl ether and collected bycentrifuging. The pellet is dissolved in 1 ml of trifluoroacetic acid,precipitated by addition of 15 ml of methyl tert-butyl ether and thenwashed with 15 ml of a methyl tert-butyl ether/petroleum ether mixture(2/1 by volume) in the presence of 0.2 ml of trifluoroacetic acid. Theproduct is dried under reduced pressure (3.5 kPa), then purified by highperformance liquid chromatography on a 100 Å C₁₈ column (250×10 mm,Bio-Rad), elution being carried out with a gradient from 20 to 40% ofacetonitrile containing 0.07% by volume of trifluoroacetic acid in watercontaining 0.07% by volume of trifluoroacetic acid at a flow rate of 6ml/minute, over 30 minutes, and then lyophilized. 3.4 mg of theditrifluoroacetate of the compound of formula (II) in which R representsan --NH--CH₂ --COOH radical, R₁ --(R₂)_(m) --(R₃)_(n) --(R₄)_(p)represents Fmoc-Arg-Arg-Pro-, R₅ and R₆ represent hydroxyl radicals andR₇ represents a hydrogen atom are thus obtained in the form of a whitepowder Mass spectrum on Sciex API III, in ESMS (Electrospray MassSpectrometry): M/z=1306 (MH)⁺, M/z=653 (M+2H)²⁺ !.

Example I

180 mg (170 μmol) of Boc-Arg(benzyloxycarbonyl)₂-Arg(benzyloxycarbonyl)-₂ -Pro-OH are treated with 31 mg (150 μmol) ofdicyclohexylcarbodiimide and 23 mg (170 μmol) of N-hydroxybenzotriazolein 3 ml of dimethylformamide for 4 hours at a temperature in the regionof 200° C. The dicyclohexylurea formed is then filtered on a 0.45 μmMillex HV filter (Millipore) and the filtrate is added to a solutioncontaining 100 mg (162 μmol) of the compound of formula (I) in which R'"represent hydrogen atoms and R, R' and R" represent hydroxyl radicalsand 0.025 ml (143 μmol) of diisopropylethylamine in 2.5 ml ofdimethylformamide. The reaction is carried out for 24 hours at atemperature in the region of 20° C. The solvent is removed by virtue ofa Speed Vac (Savant) centrifugal evaporator equipped with a vane pumpfor 18 hours at a temperature in the region of 20° C. The residue isdissolved in 20 ml of dichloromethane and the solution extractedsuccessively with 2 times 4 ml of distilled water, 2 times 4 ml of a0.1M solution of sodium dihydrogenphosphate in distilled water and 2times 4 ml of distilled water. The compound of formula (II) in which R₁--(R₂)_(m) --(R₃)_(n) --(R₄)_(p) represents Boc-Arg(benzyloxycarbonyl)₂-Arg(benzyloxycarbonyl)₂ -Pro-, R, R₅ and R₆ represent hydroxyl radicalsand R₇ represents a hydrogen atom is thus obtained in solution indichloromethane. This solution is divided into three equal parts whichare dried. One of these parts is dissolved in 2 ml of adichloromethane/methanol mixture (1/1 by volume). 100 mg of 10%palladium-on-charcoal and 100 mg of ammonium formate are added to thissolution. The reaction is carried out for 100 minutes with periodicstirring to a vortex. The reaction medium is then filtered on a 0.45 μmMillex HV filter (Millipore) and the filter washed with 5 ml ofmethanol. The solvent is evaporated under reduced pressure (2.6 kPa,450° C., 30 minutes). The product is finally purified by highperformance liquid chromatography on a 100 Å C₁₈ column (250×10 mm,Bio-Rad), elution being carried out with a gradient from 15 to 40% ofacetonitrile containing 0.07% of trifluoroacetic acid (by volume) inwater containing 0.07% of trifluoroacetic acid (by volume) at a flowrate of 6 ml/minute over 30 minutes, and then lyophilized. 15.2 mg ofthe compound of formula (I) in which R₁ --(R₂)_(m) --(R₃)_(n) --(R₄)_(p)represents Boc-Arg-Arg-Pro-, R, R₅ and R₆ represent hydroxyl radicalsand R₇ represents a hydrogen atom are thus obtained in the form of awhite powder Mass spectrum on Sciex API III, in ESMS (Electrospray MassSpectrometry): M/z=1126 (MH)⁺, M/z=563 (M+2H)²⁺ !.

The peptide Boc-Arg(benzyloxycarbonyl)₂ -Arg(benzyloxycarbonyl)₂ -Pro-OHcan be prepared in the following way: the compoundBoc-Arg(benzyloxycarbonyl)₂ -OH is reacted with methyl prolinate in thepresence of 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate in acetonitrile or dimethylformamide,diisopropylethylamine being used as base in order to obtain a pH above8, and the Boc group is then cleaved with 40% trifluoroacetic acid indichloromethane. The compound Boc-Arg(benzyloxycarbonyl)₂ -OH is reactedwith this product in the presence of2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphatein acetonitrile or dimethylformamide, diisopropylethylamine being usedas base in order to obtain a pH above 8. The ester obtained can then besaponified with lithium hydroxide at a temperature in the region of +40°C.

Example J

One of the 3 parts of the compound of formula (II) in which R₁ -(R₂)_(m)--(R₃)_(n) --(R₄)_(p) represents Boc-Arg(benzyloxycarbonyl)₂-Arg(benzyloxycarbonyl)₂ -Pro-, R, R₅ and R₆ represent hydroxyl radicalsand R₇ represents a hydrogen atom obtained in Example I is treated for90 minutes with 5 ml of a trifluoroacetic acid/distilled water mixture(95/5 by volume). The medium is then brought to dryness on a rotaryevaporator (2.6 kPa, 450° C., 45 minutes) and 15 ml of distilled waterare then added to the medium. The suspension obtained is frozen to -80°C. and then lyophilized (96 hours). The product is dissolved in 1 ml ofdimethylformamide containing 0.0625 ml of diisopropylethylamine (0.36mmol). 50 1l (50 ,mol) of a solution of 1-adamantylacetic acid chloride(obtained by reaction of 38.8 mg (200 μmol) of 1-adamantylacetic acidwith 17.2 ml (200 μmol) of oxalyl chloride in 200 μl of dichloromethanein the presence of a drop of dimethylformamide for 15 minutes at atemperature in the region of 20° C.) are then added to the peptidesolution and the reaction is carried out at a temperature in the regionof 20° C. for 1 hour. The solvent is then removed by virtue of a SpeedVac (Savant) centrifugal evaporator equipped with a vane pump for 18hours at a temperature in the region of 20° C. The residue is dissolvedin 2 ml of a dichloromethane/methanol mixture (1/1 by volume) and then100 mg of 10% palladium-on-charcoal and 100 mg of ammonium formate areadded. The reaction is carried out for 150 minutes with periodicstirring to a vortex. The reaction medium is filtered on a 0.45 μmMillex HV filter (Millipore) and the filter washed with 5 ml ofmethanol. The solvent is then evaporated under reduced pressure (2.6kPa, 450° C., 30 minutes). The product is finally purified by highperformance liquid chromatography on a 100 Å C₁₈ column (250×10 mm,Bio-Rad), elution being carried out with a gradient from 15 to 40% ofacetonitrile containing 0.07% of trifluoroacetic acid (by volume) inwater containing 0.07% of trifluoroacetic acid (by volume) at a flowrate of 6 ml/minute, over 30 minutes, and then lyophilized. 2.9 mg ofthe ditrifluoroacetate of the compound of formula (II) in which R₁--(R₂)_(m) --(R₃)_(n) --(R₄)_(p) represents1-adamantylacetyl-Arg-Arg-Pro-, R, R₅ and R₆ represent hydroxyl radicalsand R₇ represents a hydrogen atom are thus obtained in the form of awhite powder Mass spectrum on Sciex API III, in ESMS (Electrospray MassSpectrometry): M/z=601.6 (M+2H)²⁺ !.

Example K

One of the 3 parts of the compound of formula (II) in which in which R₁--(R₂)_(m) --(R₃)_(n) --(R₄)_(p) represents Boc-Arg(benzyloxycarbonyl)₂-Arg(benzyloxycarbonyl)₂ -Pro-, R, R₅ and R₆ represent hydroxyl radicalsand R₇ represents a hydrogen atom obtained in Example I is treated for90 minutes with 5 ml of a trifluoroacetic acid/distilled water mixture(95/5 by volume). The medium is then brought to dryness on a rotaryevaporator (2.6 kPa, 450° C., 45 minutes). 15 ml of distilled water areadded to the residue and the suspension is frozen to -80° C. and thenlyophilized (96 hours). The product obtained is dissolved in 1 ml ofdimethylformamide containing 62.5 μl of diisopropylethylamine (0.36mmol). 9.9 mg (50 μmol) of 1-adamantylcarboxylic acid chloride are thenadded to the peptide solution and the reaction is carried out at atemperature in the region of 200° C. for 1 hour. The solvent is thenremoved by virtue of a Speed Vac (Savant) centrifugal evaporatorequipped with a vane pump for 18 hours at a temperature in the region of20° C. The residue is dissolved in 2 ml of a dichloromethane/methanolmixture (1/1 by volume) and then 100 mg of 10% palladium-on-charcoal and100 mg of ammonium formate are added. The reaction is carried out for150 minutes with periodic stirring to a vortex. The reaction medium isthen filtered on a 0.45 μm Millex HV filter (Millipore) and the filterwashed with 5 ml of methanol. The solvent is evaporated under reducedpressure. The product is finally purified by high performance liquidchromatography on a 100 Å C₁₈ column (250×10 mm, Bio-Rad), elution beingcarried out with a gradient from 18 to 43% of acetonitrile containing0.07% of trifluoroacetic acid (by volume) in water containing 0.07% oftrifluoroacetic acid (by volume) at a flow rate of 6 ml/minute, over 30minutes, and then lyophilized. 3.1 mg of the ditrifluoroacetate of thecompound of formula (II) in which R₁ --(R₂)_(m) --(R₃)_(n) --(R₄)_(p)represents 1-adamantylcarbonyl-Arg-Arg-Pro-, R, R₅ and R₆ representhydroxyl radicals and R₇ represents a hydrogen atom are thus obtained inthe form of a white powder Mass spectrum on a Sciex API III, in ESMS(Electrospray Mass Spectrometry): M/z=594.5 (M+2H)²⁺ !.

Example L

100 mg (85 μmol) of Fmoc-Arg(Pmc)-Arg(Pmc)-Pro-OH are treated with 17.4mg of dicyclohexylcarbodiimide (85 μmol) and 12.2 mg ofN-hydroxybenzotriazole (90 μmol) in 1.5 ml of dimethylformamide for 4hours at a temperature in the region of 20° C. The dicyclohexylureaformed is then filtered on a 0.45 μm Millex HV filter (Millipore) andthe filter is washed with 0.5 ml of dimethylformamide. 0.45 ml of thefiltrate obtained (corresponding to 19 μmol ofFmoc-Arg(Pmc)-Arg(Pmc)-Pro-OH) is then added to 0.25 ml of a solutioncontaining 16.7 mg (22.5 μmol) of the compound of formula (II) in whichR, R₅ and R6 represent hydroxyl radicals and R₇ represents an iodineatom and 4 μl (23 μmol) of diisopropylethylamine in dimethylformamide.The reaction is carried out for 3 hours at a temperature in the regionof 200° C. The solvent is removed by virtue of a Speed Vac (Savant)centrifugal evaporator equipped with a vane pump for 18 hours at atemperature in the region of 200° C. The residue is then taken up in 5ml of a trifluoroacetic acid/phenol/ethanedithiol/thioanisole/watermixture (40/311/2/2 by volume) and left to react for 90 minutes at atemperature in the region of 20° C., with stirring. The mixture is thenconcentrated on an RC10-10 (Jouan) centrifugal evaporator equipped witha vane pump for 45 minutes (temperature of the evaporating chamber 50°C., trapping of the vapours at -90° C). 15 ml of a tert-butyl methylether/petroleum ether mixture (1/1 by volume) are added to theconcentrate obtained (approximately 1 ml) in order to precipitate thepeptide. The precipitate is collected by centrifuging and dissolved in 1ml of trifluoroacetic acid. The precipitation operation is repeatedonce. The peptide is dried under reduced pressure (3.5 kPa). The productis finally purified by high performance liquid chromatography on a 100 ÅC₁₈ column (250×10 mm, Bio-Rad), elution being carried out with agradient from 22 to 47% of acetonitrile containing 0.07% oftrifluoroacetic acid (by volume) in water containing 0.07%trifluoroacetic acid (by volume) at a flow rate of 6 ml/minute, over 30minutes, and then lyophilized. 10 mg of the ditrifluoroacetate of thecompound of formula (II) in which R₁ --(R₂)_(m) --(R₃)_(n) --(R₄)_(p)represents Fmoc-Arg-Arg-Pro-, R, R₅ and R₆ represent hydroxyl radicalsand R₇ represents an iodine atom are thus obtained in the form of awhite powder Mass spectrum on Sciex API III, in ESMS (Electrospray MassSpectrometry): M/z=1374, (M+H)⁺, M/z=687 (M+2H)²⁺ !.

The compound Fmoc-Arg(Pmc)-Arg(Pmc)-Pro-OH can be prepared in thefollowing way: Fmoc-Pro-chlorotrityl resin is obtained by treating 0.3mmol of chlorotrityl chloride/polystyrene-1% divinylbenzene copolymerresin (Novabiochem) with 1 mmol of Fmoc-proline in 3.5 ml of adichloromethane/diisopropylethylamine mixture (6/1 by volume) for 30minutes at a temperature in the region of 20° C. 2 ml of methanol arethen added and the reaction is left to continue for 30 minutes at atemperature in the region of 20° C. The resin is then washedsuccessively with 3 times 5 ml of methanol and 3 times 5 ml ofdichloromethane and dried.

The peptide Fmoc-Arg(Pmc)-Arg(Pmc)-Pro-OH is assembled on an AppliedBiosystems 431A device by using the "standard Fmoc" cycles provided bythe manufacturer, N-methyl-2-pyrrolidone being used as solvent.Deprotection of the α-amine functional groups is carried out by 20%piperidine in N-methyl-2-pyrrolidone for 20 minutes at each stage in thesynthesis. The Fmoc-Pro-chlorotrityl resin is deposited in the reactorof the device. After deprotection of the α-amine functional group of theproline, the N-hydroxybenzotriazolyl ester of Fmoc-Arg(Pmc) is formed byreaction of 1 mmol of Fmoc-Arg(Pmc)-OH in 2.1 ml ofN-methyl-2-pyrrolidone, 1 ml of 1M N-hydroxybenzotriazole inN-methyl-2-pyrrolidone and 1 ml of 1M dicyclohexylcarbodiimide inN-methyl-2-pyrrolidone for 20 minutes. After removal of thedicyclohexylurea formed, the ester is reacted for 30 minutes with theresin. The a-amino functional group of the arginine is then deprotectedand the N-hydroxybenzotriazolyl ester of Fmoc-Arg(Pmc)-OH is coupled tothe resin as described above. The peptide Fmoc-Arg(Pmc)-Arg(Pmc)-Pro-OHis detached from the resin by treating the latter for 15 minutes with 10ml of an acetic acid/trifluoroethanol/dichloromethane mixture (1/2/7 byvolume) at a temperature in the region of 20° C. The resin is thenwashed successively with 10 ml of dichloromethane and 10 ml ofacetonitrile. The organic phases are combined and the solvents areremoved on a rotary evaporator (2.6 kPA, 1 hour, 45° C). The residue istaken up in 1 ml of acetonitrile and 30 ml of distilled water are added.The suspension obtained is frozen to -80° C. and lyophilized. 102 mg ofFmoc-Arg(Pmc)-Arg(Pmc)-Pro-OH are thus obtained

Example M

3.06 g of the compound of formula (I) in which R, R' and R" representhydroxyl radicals and R'" represents a hydrogen atom are dissolved in 80ml of a 5% aqueous sodium carbonate solution, to which 1.8 g of9-fluorenylmethyl N-succinyl carbonate and then 40 ml of dioxane areadded. The reaction is continued for 5 hours at a temperature in theregion of 200° C. The reaction mixture is extracted with 2 times 100 mlof ethyl acetate; the organic phases are removed and the aqueous phaseis then acidified to a pH between 1 and 2 with a concentratedhydrochloric acid solution. Extraction is carried out with 3 times 100ml of ethyl acetate and the organic phases are combined, dried withsodium sulphate, filtered over silica gel and then evaporated underreduced pressure (2 kPa). The precipitate is disintegrated in 300 ml ofa methyl tert-butyl ether/methylene chloride mixture (1/1 by volume) andthen dried under reduced pressure to give 3.3 g of the compound offormula (II) in which R₁ --(R₂)_(m) --(R₃)_(n) --(R₄)_(p) representsFmoc, R, R₅ and R₆ represent hydroxyl radicals and R₇ represents ahydrogen atom in the form of a white powder Mass spectrum on Sciex APIIII, in ESMS (Electrospray Mass Spectrometry): M/z=861 (M+Na)⁺, M/z=839(M+H)⁺ ; Infrared spectrum (FMIR in methanol): 3395, 3335, 2970, 2940,2880, 3125 to 2125, 1700, 1665, 1590, 1500, 1450 and 1225; NMR spectrum(400 MHz, DMSO, Temp.=297K, δ in ppm): R-Fmoc-1: HA1, HA2 4.23, HB 4.48,HG1, HG2 7.70, HD1, HD2 7.37, HE1, HE2 7.45, HZ1, HZ2 7.92; X-TYR1: NH5.73, HA 4.52, HB1, HB2 2.93, 3.03, HD1 6.70, HD2 6.72 HE1 6.70; X-ILE2:NH 8.36, HA 4.12, HB 1.62, MG1 0.83, HG21, HG22 1.18,1.49, MD 0.83;X-TYR3: NH 8.67, HA 3.58, HB1, HB2 2.45, 2.79, HD1 6.57, HD2 5.51;X-TYR4: NH 4.17, HA 4.28, HB1, HB2 3.18, 3.43, HD1 7.19, HD2 7.31, HE17.52, HE2 6.80!.

Example N

100 mg of Fmoc-Arg(Pmc)-Arg(Pmc)-Pro-OH (85 μmol) are treated with 17.4mg of dicyclohexylcarbodiimide (85 μmol) and 12.2 mg ofN-hydroxybenzotriazole (90 μmol) in 1.5 ml of dimethylformamide for 4hours at a temperature in the region of 200° C. The dicyclohexylureaformed is then filtered on a 0.45 μm Millex HV filter (Millipore) andthe filter is washed with 0.5 ml of dimethylformamide. 0.972 ml of thefiltrate obtained (corresponding to 41 μmol ofFmoc-Arg(Pmc)-Arg(Pmc)-Pro-OH) is then added to 0.5 ml of a solutioncontaining 32 mg (48.6 μmol) of the compound of formula (I) in which R,R' and R" represent methoxy radicals and R'" represents a hydrogen atomand 7 μl (40 μmol) of diisopropylethylamine. The reaction is carried outfor 3 hours at a temperature in the region of 20° C. The solvent is thenremoved by virtue of a Speed Vac (Savant) centrifugal evaporatorequipped with a vane pump for 18 hours at a temperature in the region of20° C. The residue is then taken up in 10 ml of a trifluoroaceticacid/phenol/ethanedithiol/thioanisole/water mixture (40131112/2 byvolume) and left to react for 90 minutes at a temperature in the regionof 20° C., with stirring. The mixture is then concentrated on an RC10-10(Jouan) centrifugal evaporator equipped with a vane pump for 45 minutes(temperature of the evaporation chamber 50° C., trapping the vapours at-90° C). 30 ml of a tert-butyl methyl ether/petroleum ether mixture (1/1by volume) are added to the concentrate obtained (approximately 2 ml) inorder to precipitate the peptide. This peptide is collected bycentrifuging. It is dissolved in 2 ml of trifluoroacetic acid and theprecipitation operation is repeated once. The peptide is dried underreduced pressure (3.5 kPa). The product is finally purified by highperformance liquid chromatography on a 100 Å C₁₈ column (250×10 mm,Bio-Rad), which is eluted with a gradient from 22 to 47% acetonitrilecontaining 0.07% of trifluoroacetic acid (by volume) in water containing0.07% of trifluoroacetic acid (by volume) at a flow rate of 6 ml/minute,over 30 minutes, and then lyophilized. 23.6 mg of the ditrifluoroacetateof the compound of formula (II) in which R₁ --(R₂)_(m) --(R₃)_(n)--(R₄)_(p) represents Fmoc-Arg-Arg-Pro-, R, R₅ and R₆ represent methoxyradicals and R₇ represents a hydrogen atom are thus obtained in the formof a white powder Mass spectrum on Sciex API III, in ESMS (ElectrosprayMass Spectrometry): M/z=1290 (M+H)⁺, M/z=646 (M+2H)²⁺ !.

We claim:
 1. A purified compound of the formula: ##STR4## in which Rrepresents a hydroxyl, alkyloxy, phenylalkyloxy or --NH--CH₂ --COOHradical, R' and R" are identical and each represent a hydroxyl ormethoxy radical and R'" represents a hydrogen, bromine, chlorine oriodine atom or a nitro radical, it being understood that the alkyl andalkyloxy radicals and portions contain 1 to 4 carbon atoms in a straightor branched chain, or a salt thereof.
 2. A process for the preparationof a compound of formula (I) according to claim 1 in which R, R' and R"represent a hydroxyl radical and R'" represents a hydrogen atom,comprising fermenting A9738 actinomycetes (CBS 162.94), isolating theproduct and optionally converting the product to a salt thereof.
 3. Aprocess for the preparation of a compound of formula (I) according toclaim 1 in which R represents a methoxy or phenylalkyloxy radical,comprising esterifying a compound of formula (I) in which R represents ahydroxyl radical, isolating the product and optionally converting theproduct to a salt thereof.
 4. A process for the preparation of acompound of formula (I) according to claim 1 in which R represents an--NH--CH₂ --COOH radical, comprising reacting a compound of formula (I),in which R represents a hydroxyl radical, with glycine, isolating theproduct and optionally converting the product to a salt thereof.
 5. Aprocess for the preparation of a compound of formula (I) according toclaim 1 in which R' and R" represent methoxy radicals, comprisingreacting a compound of formula (1), in which R' and R" representhydroxyl radicals, with trimethylsilyldiazomethane, isolating theproduct and optionally converting the product to a salt thereof.
 6. Aprocess for the preparation of a compound of formula (I) according toclaim 1 in which R'" represents a nitro radical, comprising nitrating acompound of formula (I), in which R'" represents a hydrogen atom,isolating the product and optionally converting the product to a saltthereof.
 7. A process for the preparation of a compound of formula (I)according to claim 1 in which R'" represents a chlorine atom, comprisingchlorinating a compound of formula (I) in which R'" represents ahydrogen atom, isolating the product and optionally converting theproduct to a salt thereof.
 8. A process for the preparation of acompound of formula (I) according to claim 1 in which R'" represents abromine atom, comprising brominating a compound of formula (I), in whichR'" represents a hydrogen atom, isolating the product and optionallyconverting the product to a salt thereof.
 9. A process for thepreparation of a compound of formula (I) according to claim 1 in whichR'" represents an iodine atom, comprising iodinating a compound offormula (I), in which R'" represents a hydrogen atom, isolating theproduct and optionally converting the product to a salt thereof.
 10. Thepurified compound according to claim 1, provided that when R is OH, andR'" is hydrogen, then R' and R" are other than a hydroxyl radical.